This invention relates to a method for immobilizing an enzyme utilizing the specific chemical reactivity of N-halogenoamide group.
Advantages of using an enzyme after immobilization are as follows. First of all, reusing of the enzyme is possible. Secondly, it is possible to carry out a continuous enzymatic reaction in a column. Thirdly, delicate control of a reaction is possible, since it is easy to separate reaction products from the enzyme. The fourth advantage is to be able to avoid various troubles due to contamination of final products with the enzyme or impurities. Further, the stability of the enzyme is increased and consequently the enzyme is easy to utilize. Lastly, it is possible to automatically carry out the enzymatic reaction and consequently to reduce the cost for production. Thus the immobilization of an enzyme has various advantages. Many attempts have so far been made to utilize enzymes after immobilization and the methods for immobilization can be classified mainly into adsorption process, inclusion process and chemical bonding process.
The adsorption process comprises adsorbing enzymes onto the surface of an insoluble solid and immobilizing the enzymes. Its operation is simple, but the adsorbability is so weak that the adsorbed enzymes are liable to be desorbed when there are salts or substrates at high concentrations. Therefore, the adsorption process is not satisfactory in view of the essential object of the immobilization. That is, the adsorption process is applicable only when carriers have an especially strong affinity for the enzyme molecules. At present, diethylaminoethyl cellulose is used as the carrier.
In the inclusion process, enzymes are contained into polymeric gels. At present, gels of copolymers of acrylamide and N,N'-methylene-bis-acrylamide are used. Since the enzymes themselves are not chemically bonded to polymers forming the gels, the enzymes are liable to flow out if lattice size of the gels is too large. However, if the lattice size is made smaller, the rate of enzymatic reaction becomes lower. Therefore, it is an important task to adjust the lattice size.
In the chemical bonding process, enzyme molecules are immobilized to the surface of a carrier by chemical covalent bonds utilizing functional groups of the enzyme molecules. Though the degree of activity of the enzymes is slightly lowered owing to the accompanying chemical reaction, the stability of the enzymes is excellent and the loss due to washing is small. Therefore, the chemical bonding process is most effective as a method for immobilizing the enzyme. However, the process still has many problems to be solved. That is, complicated steps and expensive reagents are necessary for chemically bonding the enzyme molecules to the surface of a carrier and also severe reaction conditions are often required. From the viewpoint of reaction forms, the processes now employed can be classified mainly into the following four procedures; (A) procedure of forming peptide bonds, (B) procedure of alkylation, (C) procedure of diazo coupling, and (D) procedure of forming Schiff's base. As the procedure (A), there may be mentioned a procedure comprising converting the carrier to azide, isocyanate, carbodiimide or iminocarbonate, and then reacting the resulting derivative with amino groups of the enzymes. As the procedure (B), there may be mentioned a procedure wherein hydroxyl groups of cellulose are activated using cyanur chloride.
As the procedure (D), there may be mentioned a procedure comprising bonding aminoalkylsilane to the surfaces of porous glass granules and then forming Schiff's salt using glutaraldehyde. However, since the formation of the Schiff's salt is an equilibrium reaction, there is a disadvantage that the enzymes are gradually released as in the adsorption process.
In the conventional processes, anyway, complicated steps, expensive reagents and strict reaction conditions are necessary for activating the carrier.
The present inventors have studied a method of immobilizing enzyme by treating the enzyme with an N-halogenoamide compound.
It is well known that the N-halogenoamide compound is formed as an intermediate compound in the Hofmann's decomposition of acid amide. That is, when an amide compound is reacted with a hypohalogenoxyacid ions (OX.sup.-) under strong alkalinity, an amine is formed through an N-halogenoamide compound as shown in the following formula: ##STR1##
As to the Hofmann's decomposition, many researches have been made on the relation between the chemical structures of amide compounds and amine yields. However, no research has yet been reported as to the chemical reactivity of an N-halogenoamide compound, a reaction intermediate, in aqueous solution thereof.
The present inventors have made studies on N-halogenation reaction of various amides, and have found that an N-halogenoamide group has a low rate of conversion to the amino group under weak alkalinity and rather reacts with atomic groups containing active hydrogen, for example, --NH.sub.2, --OH, --CONH.sub.2, etc., to form ureide, urethane, and acylurea, respectively. ##STR2##
It has been found that the N-halogenoamide group is especially active upon the amino group and is very reactive even under mild reaction conditions such as pH 8 - 9 and reaction temperature of 30.degree. - 40.degree. C.